Type 1 diabetes (also known as insulin dependent diabetes mellitus, IDDM) is an autoimmune disease that results from the destruction of the beta-cells in the pancreas. Environmental factors, such as diet and bacterial antigens play an important role in the onset of the disease (Scott F W, 1996, Diabetes Metab. Rev. 12, 341-359; Scott F W et al., 2002 Diabetes 51, 73-78; Visser J. et al., 2003, Metabolism 52, 333-337).
The immune response of the gut of the neonate is less well-developed than in adults, and this relative immune deficiency makes it easier to induce tolerance to orally administered antigens. The possibility that type 1 diabetes may be related to antigens encountered via the gut lumen has been discussed since the mid-1980s, when evidence first appeared that diet could affect the spontaneous development of diabetes in BB rats and studies in patients implicated early exposure to breast milk substitutes as a risk factor.
Attempts to prevent autoimmune diseases by inducing oral tolerance to selected individual autoantigens have met with mixed success in animals and humans (Gale A. E. 2000, Lancet 356, 526-527).
The diabetogenic factor of the milk appears to be in the casein fraction, at list in the non-obese diabetic (NOD) mice, although other proteins were suggested. It was reported (Virtanen et al. 1993, Diabetes 42 1786-1790) that early introduction of dairy products is associated with increased risk of type 1 diabetes in Finnish children. The most consistently diabetogenic diet in animal models for type 1 diabetes is the commercial, cereal/plant-based rodent diet. In contrast, hydrolyzed casein (HC) diet, in which the sole protein source is hydrolyzed casein, has a protective effect. For instance, it was shown that feeding diabetes-prone bio-breeding rats (BB-DP rats), a HC-diet reduced the diabetic incidence by 30-50% (Visser J. et al., 2003 ibid). Suggested mechanisms for this effect are an actively induced protection against the development of diabetes or avoiding exposure to diabetogenic substances present in plant-based rodent diet. Scott and colleagues reported that the effect of plant-based diet on diabetes was dose-dependent, demonstrating that the diabetogenic load of the diet is important (Scott F W et al., 1997, Diabetes 46, 589-598). Moreover, Li et al. reported hyperexpression of MI-IC class I antigens on beta cells in plant-based diet fed BB-DP rats (Li X-B et al., 1995, Diabetologia 38, 1138-1147). Furthermore, BB-DP rats receiving HC diet from weaning displayed a shift from a Th1 cytokine pattern to a Th2 cytokine pattern in the pancreas at 70 days. Accordingly, when BB-DP rats were weaned on the control diet and after the age of 50 days switched to a HC diet there was an increase in Th2 cytokines in the pancreas, but interferon gamma levels were not affected. Taken together, it seems that some food ingredients can elicit a Th1-response in the pancreas which could lead to destruction of the beta cells, that is prevented by the HC diet.
Various self-antigens have been suggested to play a role in the development of diabetes. Indeed, antibodies against glutamic acid decarboxylase (GAD), insulin, islet cell antigen (ICA-69), and hsp60 have been found in the circulation at the onset of diabetes in humans (Cohen I R 2002, Diabetologia 45, 1468-1474, Yoon J W and Jun H S, 2001, Annals of the NY academy of sciences 928, 200-211), and in pre-diabetic NOD-mice (Brudzynski, 1993, Diabetes 42, 908-13.) and BB rats. Furthermore, antibodies against bacterial hsp65 cross-react with self (human or murine) hsp60. Recent reports have shown that hsp60 molecules also reside within insulin vesicles in the islets. As a result of stress heat shock proteins are synthesized in increased amounts to refold misfolded proteins.
Environmental factors were suggested in the early eighties as trigger for Type 1 Diabetes. The evidence that this environmental trigger is to be found in cow's milk is based on epidemiological (Leslie & Elliott 1994, Diabetes 43, 843-850), ecological (Virtanen et al, 1993, Diabetes 42, 1786-1790) and animal experimental evidence (Elliott & Martin, 1984, Diabetologia 26, 297-299). The diabetogenic factor of the milk appears to be in the casein fraction, at least in the non-obese diabetic (NOD) mouse. Whey protein does not appear to contain any diabetogenic component. It has been suggested that bovine serum albumin (BSA), is the diabetogenic component of cows milk. However, a review of the evidence supporting this theory does not indicate that BSA was ever tested for diabetogenic activity in the absence of β-casein.
Latent autoimmune diabetes in adults (LADA) is a special form of diabetes, which could represent a late manifestation of type 1 diabetes. The immune destructive process is much slower, making it sometimes difficult to distinguish clinically between type 1 and type 2 diabetes. The frequency of LADA patients among all patients diagnosed as type 2 varies between 6-50% in various populations. The frequency is higher in younger age groups. Most of the LADA patients will require insulin within three years. It is still unclear whether early treatment with insulin is beneficial for the remaining beta cells.
International PCT Application WO 95/10537 discloses a method of producing denatured bovine serum albumin milk products. It is stated that the consumption of denatured BSA milk products tends to reduce the likelihood of a person acquiring type 1 diabetes. However, there is no evidence presented of any trials where either human or animal subjects were fed milk or milk products with denatured BSA. It relies upon the theory mentioned above that BSA is the diabetogenic component of cows' milk.
U.S. Pat. No. 6,451,368 discloses a method for selecting non-diabetogenic milk or milk product based on the finding that specific variant of casein has diabetogenic activity while another variant does not have. The disclosure shows that antibodies to mixed caseins are found at higher levels in newly diagnosed diabetics than in normal controls.
U.S. Pat. No. 6,451,552 discloses a method for selective production of a casein/caseinate hydrolysate stripped of immunogenic proteins by treating milk with a protease that selectively hydrolyzes casein and subsequent separating the hydrolysed casein from unhydrolyzed immunogenic protein by membrane ultrafiltration. The inventors claim that the resulting hydrolyzate is essentially free of antigenic components of the ABBOS peptide and bovine serum albumin.
Elias et al. (1997, 46, 758-64.) demonstrated a specific peptide of human hsp60, p277, to be one of the immunodominant epitopes in autoimmune diabetes. Accordingly, T-cell reactivity to p277 has been reported at the onset of diabetes in NOD mice. Interestingly, subcutaneous administration of p277 downregulated T-cell reactivity to beta cell antigens and prevented the development of diabetes in NOD mice. Treatment induced p277-specific IgG1 antibodies as well as an increase in p277-specific IL-4 and IL-10 secretion and a decrease in gamma interferon secretion, suggesting an upregulation of the Th2 cytokine pathway. The destruction of the islets of Langerhans in the pancreas is believed to be a Th1 response. A shift of Th1 to Th2 response caused by p277 could be the cause of the attenuation of diabetes.
Heat shock proteins (HSPs) are highly conserved proteins expressed in all pro- and eukaryotic cells. They are involved in many important cellular processes such as correct folding of newly synthesized proteins and subunit assembly and therefore are termed molecular chaperones (Bukau, B., et al. 2000, Cell 101, 119-122). Under non-physiological conditions like high temperature, ultraviolet radiation, a viral or bacterial infection, cellular HSP synthesis is up-regulated. HSPs exert cytoprotective functions such as preventing the aggregation of denatured proteins, initiating their refolding or proteolytic degradation (Singh-Jasuja, H., et al. 2001, Biol. Chem. 382, 629-636). According to their molecular weight, HSPs are divided into six subfamilies: small HSPs, HSP40, HSP60, HSP70, HSP90 and HSP100. They are located in the cytosol (HSP70, HSP90, HSP100), in the endoplasmic reticulum (HSP70, HSP90) or in mitochondria (HSP60).
Recently, the HSP60, HSP70, and HSP90 subfamilies have attracted increasing attention because of their potential roles in immunologically relevant processes. Several studies have identified HSPs as targets of immune responses during microbial infections (Zugel, U., and Kaufmann, S. H., 1999, Immunobiology 201, 22-35). Because of the high sequence homology between microbial FISPs and endogenous HSPs derived from damaged or stressed tissue, immunological cross-reactivity was suggested to contribute to the development of autoimmune disorders including rheumatoid arthritis and diabetes (Holoshitz, J., et al. 1986, Lancet 2, 305-309; Elias, D., et al., 1991, Proc. Natl. Acad. Sci. U.S.A 88, 3088-3091; Abulafia-Lapid, R., et al., 1999, J. Autoimmun. 12, 121-129).
Hsp60 is a mitochondrial chaperone with a major role in protein folding and unfolding as well as translocation of proteins into mitochondria. Hsp60 is found in the cell cytosol under stressful and inflammatory conditions; infection or elevated cytokine levels will induce the cellular stress response. Therefore, it is not surprising that hsp60 is a highly immunogenic protein: it is the “common antigen” of gram-negative bacteria. Immunological reactivity to both bacterial and autologous-hsp60 is highly prevalent in the general population, since the pathogen-directed immune response can easily convert into an autoimmune response due to the high homology.
T-cell responses to multiple hsp60 epitopes are present in various autoimmune and inflammatory diseases including type 1 diabetes, rheumatoid and juvenile arthritis, multiple sclerosis, ankylosing spondylitis, pelvic inflammation-associated infertility, inflammatory bowel disease, atherosclerosis, graft rejection and more. The immune system reacts to hsp60 epitopes that are either cross-reactive between the human and bacterial analogues, or idiosyncratic.
Many disclosures claim uses of heat shock proteins or fragments thereof as immune modulators in diagnosis, treatment or prevention of autoimmune diseases. Most of these disclosures relate to heat shock protein 60 also known previously as hsp65, or fragments of this protein. Antibodies against the 60 kDa heat shock protein 60 (hsp60), which have a high homology to bacterial hsp65, have been found in the circulation at the onset of diabetes in humans and in pre-diabetic NOD-mice.
For example, the particular protein produced by the human body during development of type 1 diabetes, which serves as a diagnostic marker for the incipient outbreak of type. 1 diabetes, is the human heat shock protein having a size of about 65 KD (human hsp65) or an antigen cross-reactive therewith as disclosed in EP 0417271, and in U.S. Pat. Nos. 5,114,844; 5,671,848; 5,578,303 and 5,780,034. It has been disclosed that fragments of this hsp60 protein may serve as therapeutically useful entities in preventing or alleviating type 1 diabetes and host vs. graft disease (U.S. Pat. Nos. 6,180,103 and 5,993,803 and WO 96/19236, WO 97/01959 and WO 98/08536).
The peptide p277 corresponding to positions 437-460 of human Hsp60 was discovered to be one of the immunodominant epitopes in autoimmune diabetes. Its analog, denoted DiaPep277™, disclosed in U.S. Pat. No. 6,180,103 and WO 96/19236 as p277(Val6, Val11) is a synthetic peptide analog of p277, in which two cystein residues at positions 6 and 11 were replaced with Valine residues. Nowhere in the prior art it is shown that DiaPep277 can be effective after oral administration and nowhere in the prior art it was shown or suggested that administration of DiaPep277 together with hydrolysed casein diet may positively influence the progress or outcome of diabetes.
An experimental study presented by the inventors of the present application is described in Brugman et al. 2004, Diabetologia, 1331-1333.
There is an unmet need to provide orally effective compositions for prevention, delay, suppression and treatment of diabetes. The present invention fulfils this need by providing orally active fragments and analogs of hsp60 and methods for improving the protective effect of a low antigenicity diet such as a hydrolyzed casein diet.